Deposition of beta-amyloid (Abeta) in vulnerable brain regions is an invariable feature of Alzheimer's disease (AD). Abeta is formed by the regulated cleavage of the amyloid precursor protein (betaAPP). The metabolism of betaAPP has been elucidated mainly by studies in clonal cell lines transfected with human betaAPP. However, since neurons are the most abundant source of Abeta and are the most affected cell-type in AD, the study of betaAPP metabolism and Abeta generation in neurons will be important to elucidate the biological processes occuring in the brain of those afflicted by AD. Our objective is to study the endogenous processing and intracellular trafficking of Abeta and betaAPP in rodent primary neuronal cultures. Furthermore, we are investigating whether the relative utilization of processing pathways for betaAPP are regulated in a similar manner in neuronal cultures as has been observed in cell lines. Known regulators of betaAPP metabolism, such as protein phosphorylation and first messenger neurotransmitter signal transduction systems, and gonadal hormone treatment are being assessed in these cortical cultures. Using radiosequencing and metabolic labeling- mass spectrometry, we have recently discovered that variant AbetaGlu11 peptides are principal Abeta species secreted by neurons. Moreover, we have found that treatment of neurons with 17beta-estradiol or protein phosphatase inhibitors are especially effective at reducing secretion of amyloidogenic Abeta peptides. The elucidation of the molecular and cellular processes governing the metabolism of betaAPP and Abeta in neurons will provide important information in the pursuit of rational therapeutic strategies for AD.